DNA Repair

M.Prasad Naidu MSc Medical Biochemistry, Ph.D.Research Scholar

Introduction
The maintenance of the integrity of the information in DNA molecules is of utmost importance to the survival of the species . The major responsibility for the fidelity of replication resides in specific pairing of nucleotide bases .

Proper pairing is dependent upon the presence of favoured tautomers of the purine & pyrimidine nucleotides .

contd
Physiological conditions strongly favors the amino & lactam forms , the unfavored tautomers may participate in mutagenic events if they were unrepaired . The equilibrium where by one tautomer is more stable than another is only about 104 or 105 in favor of that with great stability.

The favoring of preferred tautomers & the proper base pairing could be ensured by monitoring the base pairing for 2 times .

contd
Double monitoring appear in both mammalian & bacterial systems . First monitoring occurs at the time of insertion of the deoxyribonucleoside triphosphates , & later by a follow up ,energy requiring mechanism which removes all improper bases that may occur in the newly formed strand . Unfavored tautomers occur more frequently than once in every 10 8 10 10 base pairs .

Single base alteration

contd
The mechanisms responsible for DNA repair in E .coli include the 3 to 5 exonuclease activities of one of the subunits of polymerase III complex & of the polymerase I molecule . The analogous mammalian enzymes ( & ) do not posses nuclease proofreading function.

contd
Replication errors occurs even with efficient repair system lead to the accumulation of mutations. Damage to DNA occurs by environmental , physical & chemical agents classified to 4 types .

The nature of mutations

Simple mutations: Transitions(pyrimidine-to-pyrimidine and purine-to-purine) Transversions(pyrimidine-purine and purine-to-pyrimidine) Insertions and deletions (a nucleotide or a small number of nucleotides)
point mutations: mutations that alter a single nucleotide

Abnormal regions of DNA , either from copying errors or DNA damage are replaced by 4 mechanisms

1) 2) 3) 4)

Mismatch repair , Base excision repair , Nucleotide excision repair , Double stranded break repair .

Mismatch Repair
Mismatch repair corrects errors made when DNA is copied , for example a Cytosine could be inserted opposite an A , or the polymerase could slip or stutter & insert 2 5 extra unpaired bases . Specific proteins scan the newly synthesized DNA , using adenine methylation within GATC sequence as the point of reference .

contd
The template strand is methylated & newly synthesized strand is not methylated . This difference allows the repair enzymes to identify the strand that contains the errant nucleotide which requires replacement . If a mismatch or small loop is found , a GATC endonuclease cuts the strand bearing the mutation at a site corresponding to the GATC .

contd
An exonuclease digests this strand from GATC through the mutation thus removing the faulty DNA . The above digestion can occur from either side if the defect is bracketed by 2 GATC sites . The defect is filled by normal cellular enzymes according to the base pairing rules.

In E .coli three proteins ( Mut S , Mut L & Mut H ) are rrequired for recognition of the mutation & nicking of the strand . Other cellular enzymes ligase , polymerase & SSBs remove & replace the strand .

MutS scans the DNA, & recognize the mismatch or the distortion in the DNA backbone .

Clinical importance
Faulty mismatch repair is linked to hereditary nonpolyposis colon cancer ( HNPCC ) . Genetic studies linked HNPCC in some families to a region of chromosome 2 . The gene on chromosome 2 is hMSH2 is human analogue of Mut S protein that is involved in mismatch repair . Mutations of hMSH2 account for 50 - 60 % of HNPCC .

contd
Another gene hMLH1 is associated with most other cases . hMLH1 gene is human analogue of bacterial mismatch repair gene Mut L .

Microsatellites are repeated sequences of DNA.

These repeated sequences are common, and normal. The most common microsatellite in the humans is a dinucleotide repeat of CA, which occurs tens of thousands of times across the genome .

contd
Muted hMSH2 & hMLH1 mismatch repair enzymes results in increased size of microsatellites , this must affect the function of a protein critical in surveillance of the cell cycle in these colon cells . The appearance of abnormally long or short microsatellites in an individual's DNA is referred to as microsatellite instability. Microsatellite instability (MSI) is a condition manifested by damaged DNA due to defects in the normal DNA repair process.

Base Excision Repair

This mechanism is suitable for replacement of a single base but is not effective at replacing regions of damaged DNA . The depurination of DNA which happens spontaneously due to the thermal lability of the purine N glycosidic bond , occurs at a rate of 5000 10,000 /cell / day at 37 C .

contd
Cytosine , adenine & Guanine bases in DNA spontaneously form uracil , hypoxanthine or xanthine respectively . None of the above are normal bases . N glycosylases can recognize these abnormal bases & remove the base itself from the DNA . This removal marks the site of the defect & allows an apurinic or apyimidinic endonuclease to excise the abasic sugar .

contd
The proper base is replaced by repair , DNA polymerase & the ligase returns the DNA to its original state , this series of events is called base excision repair . By similar series of steps involving initially the recognition of the defect , alkylated bases & base analogues can be removed from DNA .

Deamination C-U Depurination ----> an abasic site

Deamination of 5-mC---->T

DNA is damaged by Alkylation, Oxidation, and Radiation

Often mispair with thymine G:C A:T

Reactive oxygen species O2-, H2O2, OH

G modification (alkylation & oxidation)

Mutations are also caused by base analogs and intercalating agents

Base analogues

Base excision repair pathway

(apurinic/apyrimidinic; recognizes missing base)

Nucleotide Excision Repair

This mechanism is used to replace regions of damaged DNA up to 30 bases in length . UV light induces the formation of cyclobutane pyrimidine pyrimidine dimers .

Smoking causes formation of benzopyrene guainine adducts .

Incapable of base-pairing and cause the DNA polymerse to stop during replication

Thymine dimer by ultraviolet light

contd
Ionizing radiation , cancer chemotherapy & chemicals found in environment cause base modification , strand breaks , cross linkage between bases on opposite strand or between DNA protein & numerous other defects are repaired by this mechanism . Nucleotide excision repair is complex process involves more gene products than 2 other types of repair , essentially involves hydrolysis of 2 phosphodiester bonds on the strand containing the defect .

contd
A special excision nuclease ( exinuclease ) consisting of at least 3 sub units in E .coli & 16 polypeptides in humans . In eukaryotic cells the enzymes cut between the 3rd to 5th phosphodiester bond 3 from the lesion & on the 5 side the cut is some where between the 21st & 25th bond . Thus a fragment of 27 29 nucleotides long is exicised . After the strand is removed it is replaced by exact base pairing through the action of polymerase ( / in humans), ends are joined by DNA ligase.

3 1 4

1.UvrA and UvrB scan DNA to identify a distortion 2. UvrA leaves the complex,and UvrB melts DNA locally round the distortion 3. UvrC forms a complex with UvrB and creates nicks to the 5 side of the lesion 4. DNA helicase UvrD releases the single stranded fragment from the duplex, and DNA Pol I and ligase repair and seal the gap

Transcription coupled DNA repair: nucleotide excision repair system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template

Clinical Imporatance
Xeroderma pigmentosum is an autosomal recessive genetic disease . The clinical syndrome include marked sensitivity to sunlight ( UV rays ) with subsequent formation of multiple skin cancers & premature death . The risk of developing skin cancer is increased 1000 to 2000 fold .

contd
The inherent defect seems to involve the repair of damaged DNA , particularly thymine dimers . Cells cultured from patients with xeroderma pigmentosum exhibit low activity for the nucleotide excision repair process . Seven complementation groups have been identified using hybrid cell analysis so at least 7 gene products ( XPA XPAG ) .

contd
XPA & XPC are involved in recognition & excision .XPB & XPD are helicases & interestingly are subunits of the transcription factor TFIIH .

Double Strand Break Repair

The repair of double strand breaks is part of the physiological process of immunoglobulin gene rearrangement . It is also important mechanism for repairing damaged DNA such as occurs as result of ionizing radiation or oxidative free radical generation . Some chemotherapeutic agents destroy cells by causing double stranded breaks or preventing their repair .

contd
Two proteins are involved in the nonhomologous rejoining of a ds break . Ku , a hetero dimer of 70 & 86 kDa subunits , bind to free DNA ends & has latent ATP dependent helicase activity .

The DNA bound Ku hetero dimer recruits an unusual DNA dependent Protein kinase ( DNA PK )

contd
DNA PK has a binding site for DNA free ends & another for ds DNA just inside these ends . It allows the approximation of the 2 separated ends . The free end DNA/Ku/DNA PK complex activates the kinase activity in the later . DNA PK reciprocally phosphorylates Ku & the other DNA PK molecule on the opposing strand , in trans .

contd
DNA PK then dissociates from the DNA & Ku, resulting in activation of the Ku helicase. This results in unwinding of the 2 ends . The unwound approximated DNA forms base pairs .

The extra nucleotide tails are removed by an exonuclease & the gaps are filled and closed by DNA ligase .

Some repair enzymes are multifunctional DNA repair proteins can serve other purposes example some repair enzymes found as components of the large TFIIH complex that play a central role in gene transcription . Another component of TFIIH is involved in cell cycle regulation . Thus three critical cellular processes may be linked through use of common proteins .

Clinical importance
In patients with ataxia telangiectasia ,an autosomal recessive disease characterized by cerebellar ataxia & lymphoreticular neoplasms , in these patients there appears to exist an increased sensitivity to damage by X rays . Fanconis anemia an autosomal recessive anemia characterized by an increased frequency of cancer & by chromosomal instability , probably have defective repair of cross linking damage.